Target (protein/gene name): Octameric Enolase E.C. Number: 4.2.1.11 NCBI Gene # or RefSeq# or PBD ID: 1W6T Organism: Streptococcus Pneumoniae Etiologic Risk Group: N/A Disease Information: Pneumococcal disease is an infection caused by Streptococcus pneumoniae bacteria (“pneumococcus”). These bacteria can cause many types of illnesses, including: pneumonia (infection of the lungs), ear infections, sinus infections, meningitis (infection of the covering around the brain and spinal cord), and bacteremia (blood stream infection). Pneumococcus bacteria are spread through coughing, sneezing, and close contact with an infected person.
Essentiality: Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues. More recently, an internal nine-amino acid motif comprising residues 248 to 256 was identified with this function. We report the crystal structure of alpha-enolase from Streptococcus pneumoniae at 2.0A resolution, the first structure both of a plasminogen-binding and of an octameric alpha-enolase. While the dimer is structurally similar to other alpha-enolases, the octamer places the C-terminal lysine residues in an inaccessible, inter-dimer groove restricting the C-terminal lysine residues to a role in folding and oligomerization. The nine residue plasminogen-binding motif, by contrast, is exposed on the octamer surface revealing this as the primary site of interaction between alpha-enolase and plasminogen.
Pairwise alignment of BlastP: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Enolase is a multifunctional glycolytic enzyme that catalyzes the conversion of D-2-phosphoglycerate to phospho(enol)pyruvate (PEP) and water. It can function as a plasminogen receptor in endothelial, epithelial, and hematopoietic cells and hence, may be involved in fibrinolytic and intravascular systems. It is also known to act as a heat shock protein, which may have implications in transcriptional and pathological functions. Enolase has been implicated in autoimmune and systemic diseases.1 Furthermore; serum neuronspecific enolase is known to function as a predictor of patient outcome post cardiac arrest.2 Thus enolase assays can be used for studying cellular functions like carbohydrate metabolism, transcription and other pathophysiological processes. The Enolase Activity Assay Kit provides a simple and sensitive procedure for measuring enolase activity in a variety of samples. Enolase activity is determined by a coupled enzyme assay in which D-2-phosphoglycerates converted to PEP, resulting in the formation of an intermediate that reacts with a peroxidase substrate, generating a colorimetric (570 nm) or fluorometric (lex = 535/lem = 587 nm) product proportional to the enolase activity present. One milliunit of enolase is the amount of enzyme that will generate 1.0 nmole of H2O2 per minute at pH 7.2 at 25 °C. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/2/mak178bul.pdf Current Inhibitors: 105 Expression Information (has it been expressed in bacterial cells):
expressed in Escherichia coli BL21(DE3)pLysS using the pRSETA expression vector, recombinant His-tagged protein Purification Method: Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MRGSHHHHHHMSIITDVYAREVLDSRGNPTLEVEVYTESGAFGRGMVPSGASTGEHEAVELRDGDKSRYGGLGTQKAVDN
VNNIIAEAIIGYDVRDQQAIDRAMIALDGTPNKGKLGANAILGVSIAVARAAADYLEIPLYSYLGGFNTKVLPTPMMNII
NGGSHSDAPIAFQEFMILPVGAPTFKEALRYGAEIFHALKKILKSRGLETAVGDEGGFAPRFEGTEDGVETILAAIEAAG
YVPGKDVFLGFDCASSEFYDKERKVYDYTKFEGEGAAVRTSAEQIDYLEELVNKYPIITIEDGMDENDWDGWKALTERLG
KKVQLVGDDFFVTNTDYLARGIQEGAANSILIKVNQIGTLTETFEAIEMAKEAGYTAVVSHRSGETEDSTIADIAVATNA
GQIKTGSLSRTDRIAKYNQLLRIEDQLGEVAEYRGLKSFYNLKK
E.C. Number: 4.2.1.11
NCBI Gene # or RefSeq# or PBD ID: 1W6T
Organism: Streptococcus Pneumoniae
Etiologic Risk Group: N/A
Disease Information: Pneumococcal disease is an infection caused by Streptococcus pneumoniae bacteria (“pneumococcus”). These bacteria can cause many types of illnesses, including: pneumonia (infection of the lungs), ear infections, sinus infections, meningitis (infection of the covering around the brain and spinal cord), and bacteremia (blood stream infection). Pneumococcus bacteria are spread through coughing, sneezing, and close contact with an infected person.
https://wwwnc.cdc.gov/travel/diseases/pneumococcal-disease-streptococcus-pneumoniae
Essentiality:
Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues. More recently, an internal nine-amino acid motif comprising residues 248 to 256 was identified with this function. We report the crystal structure of alpha-enolase from Streptococcus pneumoniae at 2.0A resolution, the first structure both of a plasminogen-binding and of an octameric alpha-enolase. While the dimer is structurally similar to other alpha-enolases, the octamer places the C-terminal lysine residues in an inaccessible, inter-dimer groove restricting the C-terminal lysine residues to a role in folding and oligomerization. The nine residue plasminogen-binding motif, by contrast, is exposed on the octamer surface revealing this as the primary site of interaction between alpha-enolase and plasminogen.
https://www.sciencedirect.com/science/article/pii/S0022283604010885?via%3Dihub
Protein Image:
Pairwise alignment of BlastP:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasite's invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.
http://www.brenda-enzymes.org/literature.php?e=4.2.1.11&r=714875
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=4.2.1.11
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Enolase is a multifunctional glycolytic enzyme that catalyzes the conversion of D-2-phosphoglycerate to phospho(enol)pyruvate (PEP) and water. It can function as a plasminogen receptor in endothelial, epithelial, and hematopoietic cells and hence, may be involved in fibrinolytic and intravascular systems. It is also known to act as a heat shock protein, which may have implications in transcriptional and pathological functions. Enolase has been implicated in autoimmune and systemic diseases.1 Furthermore; serum neuronspecific enolase is known to function as a predictor of patient outcome post cardiac arrest.2 Thus enolase assays can be used for studying cellular functions like carbohydrate metabolism, transcription and other pathophysiological processes. The Enolase Activity Assay Kit provides a simple and sensitive procedure for measuring enolase activity in a variety of samples. Enolase activity is determined by a coupled enzyme assay in which D-2-phosphoglycerates converted to PEP, resulting in the formation of an intermediate that reacts with a peroxidase substrate, generating a colorimetric (570 nm) or fluorometric (lex = 535/lem = 587 nm) product proportional to the enolase activity present. One milliunit of enolase is the amount of enzyme that will generate 1.0 nmole of H2O2 per minute at pH 7.2 at 25 °C.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/2/mak178bul.pdf
Current Inhibitors: 105
Expression Information (has it been expressed in bacterial cells):
expressed in Escherichia coli BL21(DE3)pLysS using the pRSETA expression vector, recombinant His-tagged protein
Purification Method: Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.
http://www.brenda-enzymes.org/literature.php?e=4.2.1.11&r=652761
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MRGSHHHHHHMSIITDVYAREVLDSRGNPTLEVEVYTESGAFGRGMVPSGASTGEHEAVELRDGDKSRYGGLGTQKAVDN
VNNIIAEAIIGYDVRDQQAIDRAMIALDGTPNKGKLGANAILGVSIAVARAAADYLEIPLYSYLGGFNTKVLPTPMMNII
NGGSHSDAPIAFQEFMILPVGAPTFKEALRYGAEIFHALKKILKSRGLETAVGDEGGFAPRFEGTEDGVETILAAIEAAG
YVPGKDVFLGFDCASSEFYDKERKVYDYTKFEGEGAAVRTSAEQIDYLEELVNKYPIITIEDGMDENDWDGWKALTERLG
KKVQLVGDDFFVTNTDYLARGIQEGAANSILIKVNQIGTLTETFEAIEMAKEAGYTAVVSHRSGETEDSTIADIAVATNA
GQIKTGSLSRTDRIAKYNQLLRIEDQLGEVAEYRGLKSFYNLKK
*length of your protein in Amino Acids: 444
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 48357.29 kDA
Molar Extinction coefficient of your protein at 280 nm wavelength: 39310 M-1cm-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Gene Available:
https://www.atcc.org/Search_Results.aspx?dsNav=Ntk:PrimarySearch%7cStreptococcus+Pneumoniae%7c3%7c,Ny:True,Ro:0,N:1000552&searchTerms=Streptococcus+Pneumoniae&redir=1
CDS Gene Sequence:
ATGTCAATTATTACTGATGTTTACGCTCGCGAAGTCCTAGACTCACGCGGTAACCCAACACTTGAAGTAG
AAGTTTACACTGAATCAGGTGCTTTCGGACGTGGTATGGTTCCATCAGGAGCTTCTACTGGTGAACACGA
AGCAGTTGAACTTCGCGACGGTGACAAATCTCGTTACGGTGGTCTTGGTACACAAAAAGCTGTTGACAAC
GTAAACAACATCATTGCTGAAGCTATCATTGGCTACGATGTACGTGATCAACAAGCTATTGACCGTGCTA
TGATCGCACTTGACGGTACTCCTAACAAAGGTAAATTGGGTGCGAATGCAATCCTCGGTGTGTCTATCG
TGTAGCTCGTGCTGCTGCTGACTACCTTGAAATCCCACTTTACAGCTACCTTGGTGGATTCAACACTAAA
GTTCTTCCAACTCCAATGATGAACATCATCAACGGTGGTTCTCACTCTGACGCTCCAATCGCTTTCCAAG
AGTTCATGATCTTGCCAGTTGGTGCGCCAACATTTAAAGAAGCCCTTCGTTACGGTGCTGAAATCTTCCA
CGCTCTTAAGAAAATCCTTAAATCACGTGGTTTGGAAACTGCCGTAGGTGACGAAGGTGGATTCGCTCCT
CGTTTCGAAGGAACTGAAGATGGTGTTGAAACTATCCTTGCTGCGATTGAAGCTGCTGGATATGTACCAG
GTAAAGACGTATTTCTCGGATTTGACTGTGCTTCATCAGAATTCTACGATAAAGAACGTAAAGTTTACGA
CTACACTAAATTTGAAGGCGAAGGTGCTGCTGTTCGTACATCTGCAGAACAAATCGACTACCTTGAAGAA
TTGGTTAACAAATACCCAATCATCACTATTGAAGATGGTATGGATGAAAACGACTGGGATGGTTGGAAAG
CTCTTACTGAACGTCTTGGTAAGAAAGTACAACTTGTTGGTGACGACTTCTTCGTAACAAACACTGACTA
CCTTGCACGTGGTATCCAAGAAGGTGCTGCTAACTCAATCCTTATCAAAGTTAACCAAATCGGTACTCTT
ACTGAAACTTTTGAAGCTATCGAAATGGCTAAAGAAGCTGGTTACACTGCTGTTGTATCACACCGTTCAG
GTGAAACTGAAGATTCAACAATCGCTGATATCGCAGTTGCAACTAACGCAGGACAAATCAAGACTGGTTC
ACTTTCACGTACAGACCGTATCGCTAAATACAACCAATTGCTTCGTATCGAAGACCAACTTGGTGAAGTA
GCTGAATATCGTGGATTGAAATCATTCTACAACCTTAAAAAATAA